Polynucleotide and polypeptide sequence and methods thereof

ABSTRACT

The present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably  Pichia pastoris.  The present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins.

TECHNICAL FIELD

The present disclosure relates to a field of recombinant DNAtherapeutics. It involves the bio-informatics design, synthesis ofartificial gene for human insulin precursor including leader peptidecoding sequence, cloning in an expression vector and expression in anorganism, preferably Pichia pastoris. The present disclosure alsorelates to methods of downstream processing for obtaining proteinprecursor molecules and subsequent conversion of precursor molecules tofunctional proteins.

BACKGROUND OF THE DISCLOSURE

Human Insulin is a polypeptide hormone involved in regulation of glucosein blood and body fluids. Production deficiency leads to type 1 or type2 diabetes. Type 1 is especially Insulin dependent diabetes. Earlier,Insulin used to be supplemented from animal sources (bovine and pig),which often results in undesirable allergic immune response orhypersensitive reaction on continual administration for longer periods.The next generation of Humanized Insulin was produced in E. coli byrecombinant DNA technology and is being successfully used for the pastseveral years. Although recombinant human Insulin is being expressed indifferent hosts through patented processes to meet the diabetestherapeutic requirements, the demand is growing and forcing the man kindto explore new and modified methods to produce commercially viablequantities.

Recombinant human Insulin currently available in the market is producedfrom at least three different expression systems i.e. E. coli, Pichiapastoris and Hansenula polymorpha. Over expression in E. coli results inproteins accumulating as insoluble inclusion bodies. Solubilization andrefolding of the recombinant Insulin from the inclusion bodies requiresuse of chaotropic chemicals such as guanidine hydrochloride, urea, etc.and presence of traces of these chemicals in the final product evenafter extensive purification could be hazardous. Alternatively, proteinscan be expressed in yeast system and secreted out into the medium atmuch higher levels in soluble form. However, levels of expressionobtained in each yeast system differed from protein to protein forunknown reasons.

The two chains of Human Insulin are also being expressed separatelyusing two different vectors and assembled together in-vitro afterpurification. Disulphide linkages between two chains is facilitated bychemical methods

STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to a polynucleotide sequenceas set forth in SEQ ID NO: 2; a polypeptide sequence as set forth in SEQID NO: 1; a method for obtaining recombinant insulin precursor moleculehaving polypeptide sequence as set forth in SEQ ID NO: 1, said methodcomprising steps of: a) synthesizing a polynucleotide sequence set forthin SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28by assembly PCR, and inserting the synthesized sequence in a vector, b)transforming a host cell with said vector followed by antibioticscreening host selection, and c) fermenting the selected transformedhost cell and in-situ capturing of the insulin precursor molecule toobtain said precursor having polypeptide sequence as set forth in SEQ IDNO: 1; a method of downstream processing for in-situ capturing ofprotein precursor molecule during fermentation process, said methodcomprising steps of: a) simultaneous pumping of fermentation productobtained during fermentation into a hollow fibre harvesting system toobtain permeate and retentate, b) recycling of the retentate into thefermentor, and c) subjecting the permeate through ion-exchangechromatographic column followed by washing with TRIS elution buffer toobtain said protein precursor molecule; a method of downstreamprocessing for in-situ conversion of protein precursor molecule intofunctional protein molecule, said method comprising step of: a)concentrating the precursor molecule through TFF Cassette and mixing theconcentrate with organic solution to obtain retentate reaction mixture,b) subjecting the reaction mixture to incubation through TPCK trypsinimmobilized column to obtain protein ester, and c) subjecting the esterto deblocking buffer followed by hydrophobic interaction chromatographiccolumn to obtain said functional protein molecule; a method forobtaining recombinant insulin molecule from a precursor molecule havingpolypeptide sequence as set forth in SEQ ID NO: 1, said methodcomprising steps of: a) synthesizing a polynucleotide sequence set forthin SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28by assembly PCR, b) inserting the synthesized sequence in a vector andtransforming a host cell with said vector followed by antibioticscreening host selection, c) fermenting the selected transformed hostcell followed by downstream processing for in-situ capturing of theinsulin precursor molecule, and d) in-situ conversion of insulinprecursor molecule having polypeptide sequence as set forth in SEQ IDNO: 1 into said recombinant insulin molecule; a recombinant vectorcomprising the polynucleotide sequence set forth in SEQ ID NO: 2; and arecombinant host cell, transformed by introduction of a vectorcomprising polynucleotide sequence set forth in SEQ ID NO: 2;

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

FIG. 01: Flow chart of cloning process—construction of insert,preparation of vector for ligation, ligation of insert with vector,preparation of vector for Pichia transformation.

FIG. 02: Agarose gel image of gene construct from Oligos by AssemblyPCR.

Lane 1: 100 base pair DNA ladder and Lanes 2-5: Assembled gene amplified(Amplicon size 640 by along with AOX1 region)

FIG. 2 a: Gel Electrophoresis—The product of second PCR is checked on 1%agarose gel in TAE buffer

1% agarose gel castingLoading—1.5 μl sucrose dye+5 μl-10 μl PCR productMarker—100 bp marker 0.8-1.0 μl +2 μl milliQ+1.5 dyePCR amplicon size is 483 and is seen on gel around 500 bp

FIG. 03: Double enzyme digestion of pPIC9K vector with BamHl and Notl

Lane 1: 1 kb DNA ladderLane 2: pPIC9K before digestionLane 3: pPIC9K after digestion

FIG. 04: Double enzyme digestion of target gene with BamHl and Notl

Lane 1: 100 bpb DNA ladderLane 2: gene double digest

FIG. 05: DNA sequencing profile with designed and sequenced codons andaminoacid details

FIG. 06: SDS-PAGE (15%) image of fermentation samples during differentstages of induction showing continuous increase of expression andsecretion of Insulin precursor into the fermentation medium.

Lane 1: Standard PIP

Lane 2: Protein molecular weight marker;Lane 3: Fermentation sample at 0 hour of induction;Lane 4: Fermentation sample at 6 hour of induction;Lane 5: Fermentation sample at 12 hour of induction;Lane 6: Fermentation sample at 24 hour of induction;Lane 7: Fermentation sample at 30 hour of induction; LaneLane 8: Fermentation sample at 36 hour of induction andLane 9: Fermentation sample at 42 hour of induction

FIG. 07: HPLC profile of Fermentation samples at different periods ofinduction. [quantity of insulin precursor Vs Time course of fermentationinduction phase].

There is a progressive increase in the quantity of precursor from timeto time of induction period. The increase in quantity is correlated withsize of peaks expressed as milli volts.

FIG. 08: Comparative HPLC profile of standard precursor and precursor infermentation (final) sample, showing selective capturing of InsulinPrecursor. The peak corresponding to standard obtained from proteinconcentration of 1 mg/mL. The peak corresponding to fermentation sampleis obtained from 1:1 dilution of broth. 50% diluted FMN broth peak ismore than that of standard precursor. It corresponds ≧2 g/lit.

FIG. 09: HPLC profile of enzymatic conversion of Insulin Precursor (PIP)to Human Insulin butyl ester (Transpeptidation Product). BeforeTranspeptidation reaction, said PIP was loaded. After trypsin digestionand transpeptidation, the product (HI ester) is also loaded onto HPLC toconfirm completion of the reaction. The mass balance is almost matchedbetween precursor and its Transpeptidation product.

FIG. 10: The TP product is deblocked to obtain Human insulin and loadedonto HPLC to know the purity and profile. In terms of mass balance it ismatching with PIP (precursor). Both PIP in FIG. 09 and HI in this figureare loaded at the same concentration (1 mg/mL). (Purified Human InsulinHPLC profile as per British Pharmacopeia 2007).

FIG. 11: Diagram showing the flow chart of fermentation, in-situharvesting and clarification of fermentation broth by a hollow fibreharvesting system with 0.2 μM cassette. The cells retained afterharvesting are directed back into fermenter along with fresh medium.

FIG. 12: Diagram shows the flowchart of concentration and in-situcapturing of human insulin precursor in cation exchange chromatographycolumn followed by in-situ digestion and transeptidation in TPCK trypsinimmobilized column for conversion into insulin butyl ester. Humaninsulin ester is deblocked and passed through HIC column to get purifiedhuman insulin.

FIG. 13: SDS PAGE (15%) of purified Human Insulin and comparison withcommercial formulation;

Lane 1: Insulin Precursor;

Lane 2: protein molecular weight marker;

Lane 3: Purified Insulin Precursor;

Lane 4: Human insulin after deblocking;Lane 5: Human insulin after polishing andLane 6: Commercial recombinant Human Insulin (Huminsulin R)

FIG. 14: Western Blot image of Insulin precursor and purified HI andcommercial HI

Lane 1: Prestained protein markerLane 2: Insulin precursor captured from FMN brothLane 3: Purified bigtec human InsulinLane 4: Commercial human insulin

FIG. 15: Codon Pair Optimization: Oligo assembled sequence (product ofassembly-PCR)

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to a polynucleotide sequence as set forthin SEQ ID NO: 2.

In an embodiment of the present disclosure, the polynucleotide encodes afusion polypeptide comprising recombinant Human Insulin Precursor andsignal peptide.

The present disclosure relates to a polypeptide sequence as set forth inSEQ ID NO: 1. In an embodiment of the present disclosure, thepolypeptide is a fusion polypeptide comprising recombinant Human InsulinPrecursor and signal peptide.

In another embodiment of the present disclosure, the polypeptidesequence corresponds to polynucleotide sequence set forth in SEQ ID NO:2, wherein the polynucleotide is subjected to post-transcriptionalmodification and codon optimization to obtain corresponding polypeptideof SEQ ID NO: 1.

The present disclosure relates to a method for obtaining recombinantinsulin precursor molecule having polypeptide sequence as set forth inSEQ ID NO: 1, said method comprising steps of:

-   -   a) synthesizing a polynucleotide sequence set forth in SEQ ID        NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by        assembly PCR, and inserting the synthesized sequence in a        vector,    -   b) transforming a host cell with said vector followed by        antibiotic screening host selection, and    -   c) fermenting the selected transformed host cell and in-situ        capturing of the insulin precursor molecule to obtain said        precursor having polypeptide sequence as set forth in SEQ ID NO:        1.

In an embodiment of the present disclosure, the polypeptide is a fusionpolypeptide comprising recombinant Human Insulin Precursor and signalpeptide.

In another embodiment of the present disclosure, the synthesizedpolynucleotide and the vector are subjected to restriction enzymedigestion for insertion of the polynucleotide into the expression vectorand wherein restriction enzymes are selected from a group comprisingBamHI, NotI, SacI, BgIII and SacII or any combination thereof.

In yet another embodiment of the present disclosure, the vector isselected from a group comprising pPIC9K and pPICZα, preferably pPIC9Kand wherein the host is selected from a group comprising Pichiapastoris, Pichia methanolica; Pichia guilliermondii and Pichiacaribbica, preferably Pichia pastoris.

In still another embodiment of the present disclosure, the in-situcapturing of the precursor molecule is carried out by hollow fibreharvesting system and ion-exchange chromatographic column to obtain saidprecursor.

The present disclosure relates to a method of downstream processing forin-situ capturing of protein precursor molecule during fermentationprocess, said method comprising steps of:

-   -   a) simultaneous pumping of fermentation product obtained during        fermentation into a hollow fibre harvesting system to obtain        permeate and retentate,    -   b) recycling of the retentate into the fermentor, and    -   c) subjecting the permeate through ion-exchange chromatographic        column followed by washing with TRIS elution buffer to obtain        said protein precursor molecule.

In an embodiment of the present disclosure, the permeate comprise ofclarified cell free broth and the retentate comprise of concentratedcells.

In another embodiment of the present disclosure, the wherein theretentate is recycled back to fermentor vessel along with fresh mediumin the fermentor and the permeate is passed through the ion-exchangechromatographic column for capturing the protein precursor.

In yet another embodiment of the present disclosure, the proteinprecursor selectively binds to polymer matrix of the ion-exchangechromatographic column and is eluted with the elution buffer.

The present disclosure relates to a method of downstream processing forin-situ conversion of protein precursor molecule into functional proteinmolecule, said method comprising step of:

-   -   a) concentrating the precursor molecule through TFF Cassette and        mixing the concentrate with organic solution to obtain retentate        reaction mixture,    -   b) subjecting the reaction mixture to incubation through TPCK        trypsin immobilized column to obtain protein ester, and    -   c) subjecting the ester to deblocking buffer followed by        hydrophobic interaction chromatographic column to obtain said        functional protein molecule.

In an embodiment of the present disclosure, the precursor molecule isconcentrated to a range of about 100 mg/ml to about 200 mg/ml.

In another embodiment of the present disclosure, the organic solutioncomprise of O-tert-Butyl-L-theronine tert-butyl ester acetate dissolvedin 1:1 v/v dimethyl sulfoxide (DMSO):Methanol and the deblocking buffercomprise a combination of tryptophan and trifluoroacetic acid.

In another embodiment of the present disclosure, the TPCK column isequilibrated with a combination of CaCl₂ and Acetic acid and thehydrophobic interaction chromatographic column is equilibrated with acombination of Acetic acid and Ammonium sulphate.

In another embodiment of the present disclosure, time for the incubationranges from about 1.5 hrs to about 3.5 hrs and temperature for theincubation ranges from about 15° C. to about 25° C.

The present disclosure relates to a method for obtaining recombinantinsulin molecule from a precursor molecule having polypeptide sequenceas set forth in SEQ ID NO: 1, said method comprising steps of:

-   -   a) synthesizing a polynucleotide sequence set forth in SEQ ID        NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by        assembly PCR,    -   b) inserting the synthesized sequence in a vector and        transforming a host cell with said vector followed by antibiotic        screening host selection,    -   c) fermenting the selected transformed host cell followed by        downstream processing for in-situ capturing of the insulin        precursor molecule, and    -   d) in-situ conversion of insulin precursor molecule having        polypeptide sequence as set forth in SEQ ID NO: 1 into said        recombinant insulin molecule.

In an embodiment of the present disclosure, the polypeptide is a fusionpolypeptide comprising recombinant Human Insulin Precursor and signalpeptide.

In another embodiment of the present disclosure, the synthesizedpolynucleotide and the vector are subjected to restriction enzymedigestion for insertion of the polynucleotide into the expression vectorand wherein the restriction enzymes are selected from a group comprisingBamHI, NotI, SacI, BgIII and SacII or any combination thereof.

In yet another embodiment of the present disclosure, the vector isselected from a group comprising pPIC9K and pPICZα, preferably pPIC9Kand wherein the host is selected from a group comprising Pichiapastoris, Pichia methanolica, Pichia guilliermondii and Pichiacaribbica, preferably Pichia pastoris.

In still another embodiment of the present disclosure, the in-situcapturing of the precursor molecule is carried out by hollow fibreharvesting system and ion-exchange chromatographic column to obtain saidprecursor.

In still another embodiment of the present disclosure, the in-situconversion of the precursor molecule is carried out by subjecting theprecursor molecule to TFF Cassette and TPCK trypsin immobilized columnto obtain protein ester.

In still another embodiment of the present disclosure, the protein esteris subjected to deblocking buffer followed by hydrophobic interactionchromatographic column to obtain said recombinant insulin molecule.

The present disclosure relates to a recombinant vector comprisingpolynucleotide sequence set forth in SEQ ID NO: 2.

In an embodiment of the present disclosure, the vector is selected froma group comprising pPIC9K and pPICZα, preferably pPIC9K.

The present disclosure relates to a recombinant host cell, transformedby introduction of a vector comprising polynucleotide sequence set forthin SEQ ID NO: 2.

In an embodiment of the present disclosure, the host is selected from agroup comprising Pichia pastoris, Pichia methanolica, Pichiaguilliermondii and Pichia caribbica, preferably Pichia pastoris andwherein the vector is selected from a group comprising pPIC9K andpPICZα, preferably pPIC9K.

The main object of the present disclosure is to de novo design andexpress the gene coding for “secretion signal and recombinant InsulinPrecursor fusion protein” comprising the amino acid sequence as setforth in SEQ ID NO: 1.

The present disclosure relates to a method for obtaining recombinanthuman insulin, said method comprising the steps of:

-   -   a) Designing and constructing an insulin precursor-signal        peptide fusion protein coding gene;    -   b) Ligating the precursor-signal peptide fusion protein coding        gene to a vector;    -   c) Obtaining multiple copies of the precursor-signal peptide        fusion protein coding gene by transformation (electroporation)        into a host.    -   d) Fermentation of transformed host cell lines to obtain        recombinant human insulin precursor; and    -   e) Obtaining the recombinant human insulin by efficient        downstream processing of the human insulin precursor.

In the present disclosure optimization of nucleotide sequence was donefor enhanced expression and secretion of target protein intofermentation medium.

Optimization at multiple steps during clone construction, cloning,transformation, fermentation, downstream processing resulted in overallincrease in yield, scalability and biological efficacy. Down-streamprocessing is improvised with, in-situ capturing and in-situ conversionof precursor to final product to minimize the processing time, cost,manpower and conserve reagents.

In another embodiment of the present disclosure, said SEQ ID No. 2 isobtained by multiple stages in-silico optimization of nucleotidesequence based on “Codon-Pair Frequency” of highly expressed proteins inPichia pastoris. The sequence was further tuned to enhance proteinsynthesis by mRNA secondary structure prediction and removing highmelting stem loop structures, which enables un-restricted ribosomemovement and high speed protein synthesis.

Codon Pair Optimization

Codon optimisation is a method of gene optimisation, where in thesynthetic gene sequence is modified to match the “codon usage pattern”for a particular organism. Here, for a particular amino acid sequence,select “most frequently used codons” (from a list of degenarate codonsfor an aminoacid), by that organism. So that the aminoacid sequenceremains same but with a different DNA sequence, matched for thatorganism. How-ever this does not consider the fact that codons are readby ribosomes in “pairs”, during protein synthesis. There are 2 codonbinding site in ribosome, on adjescent places. Extensive analysis wasdone and a particular pattern was observed in which the “codon-pairs”are used by pichia pastoris. So the construct DNA sequence was modifiedto match to this “codon-pair usage frequency”. This methodology is noveland never reported for gene expression optimisation. A proprietaryin-house developed software was used for this excercise. By doing thisgene optimisation (FIG. 15), it was found that the expression levelcould be increased by approx 30%.

In another embodiment of the present disclosure, the whole gene sequencei.e. Insulin Precursor and secretion signal was subjected tooptimization together, as it is expressed as a single chain protein inthe expression host.

In yet another embodiment of the present disclosure, said precursor isconstructed with about 26 oligonucleotides coding for Insulinprecursor-Signal peptide fusion protein.

In yet another embodiment of the present disclosure, said vector isselected from a group comprising pPIC9K and pPICZα, preferably pPIC9K

In still another embodiment of the present disclosure, said cloning iscarried out at downstream of AOX1 promoter in pPIC9K vector.

In still another embodiment of the present disclosure, said host isselected from a group comprising Pichia pastoris, Pichia methanolica,Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris.

In still another embodiment of the present disclosure, said cloning wascarried out by simultaneous multiple gene insertions and directselection using an antibiotic to get high copy number of gene into thehost

In still another embodiment of the present disclosure, said fermentationis carried out in a modified low salt minimal medium at optimaltemperature range, aeration, cell densities and feeding, which enableshigh level expression and easy downstream processing.

In still another embodiment of the present disclosure, fermentationprocess and harvesting process are coupled. It involves a hollow fibreharvesting module is connected to fermenter for in-situ filtration ofbroth during harvesting. The culture from fermenter is pumped to ahollow fibre cassette to separate cell free broth from the cells. Thecells after filtration are recycled back to fermenter vessel along withmedium to maintain culture volume and promote normal growth of culture.

In still another embodiment of the present disclosure capturing of humaninsulin precursor is coupled with trypsin digestion and transpeptidationin an immobilized trypsin column. It involves binding of insulinprecursor in cell free broth from hollow fibre filtration system to achromatography column packed with high binding capacity synthetic resin.The unbound is again channeled back into fermenter along with freshmedium.

The bound protein is eluted and further channeled into a column packedwith TPCK trypsin immobilized to matrix. On the way to Trypsin columnthe eluted precursor is mixed with necessary buffers and desired PH. Theprecursor is converted into insulin ester by tryptic digestion andtranspeptidation in the column. Then the insulin ester is eluted fromcolumn and deblocked to convert into human insulin and lyophilized.Finally the human insulin is polished to highest purity by reverse phasechromatography.

In still another embodiment of the present disclosure, said fermentationmedium has a pH ranging from about 4.0-5.0, preferably about 4.75 duringinitial phase of fermentation; about 4.0-5.0, preferably about 4.80during glycerol phase and about 4.0-5.0, preferably about 4.95 duringinduction phase.

In still another embodiment of the present disclosure, said temperatureat fermentation ranges from about 29-30° C., preferably about 30.0° C.for batch phase; about 29-30° C., preferably about 29.5° C. for glycerolfed batch; and about 27-29° C., preferably about 28.0° C. for inductionphase with methanol.

In still another embodiment of the present disclosure, said aeration atfermentation ranges from about 0.5-1.5 VVM pure air, preferably 1VVMpure air for batch phase; about 0.5-1.5 VVM air:oxygen, preferably about1.0 VVM air: oxygen (about 90:10) for glycerol batch; and about 1.5 VVMair:oxygen ratio begins at about 85:15 and ends at about 40:60 with anincrement/decrement of about 5 at about every 5 hours for methanol batch(induction phase).

In still another embodiment of the present disclosure, duringfermentation glycerol feeding is carried out to promote high celldensity growth before induction and is continued until cell density(OD₆₀₀) reaches about 500. Then methanol is fed exponentially to promoteincreased expression of target protein.

In the present disclosure, a synthetic gene having modified nucleotidesequences and coding for a gene comprising the Mat-α secretion signal,spacer, and the insulin precursor was designed de novo. Extensivebioinformatics analysis was used to arrive at a novel coding sequence,based on nucleotide patterns from highly expressed proteins in Pichiapastoris. The synthetic gene (482 bp) was constructed by synthesizing 26oligonucleotides and combining them by assembly PCR.

Pichia expression system is known for its very high levels ofexpression, using a methanol inducible promoter. Proteins can beexpressed as secretory proteins and therefore purification of the samebecomes simple. The doubling time of the strain, ease of handling,minimal growth requirements, availability of convenient vectors, hostsystems and selection methodologies make Pichia pastoris an ideal andattractive system for study. High cell densities are achievable inminimal mineral media and the ease of induced expression of proteinsadds to the convenience of using this system for recombinant proteinexpression.

The insulin precursor fusion protein gene obtained by assembly PCR wasconfirmed by DNA sequencing (FIG. 05) and was cloned into Pichiapastoris expression vector pPIC9K. The vector after linearizationtransformed into GS115 strain of Pichia by electroporation. Theexpression cassette was integrated into the Pichia host system byhomologous recombination. Clones harboring high copy number inserts werepicked by antibiotic screening. Clones showing maximum resistance to theantibiotic genticin (G418) were picked and screened for their ability toexpress and secrete the Insulin precursor into the culture medium.Promising clones were further evaluated by 7 liter capacity fermenter.The fermentation yield of insulin precursor is around 1.5 gm/litre. Thiscan be further increased through additional optimization of the process.

The secreted insulin precursor was captured from the broth, purified andenzymatically modified to obtain Human Insulin. Biological activity ofthe final product in terms of regulating blood glucose has beenestablished in mice and rats and found to be comparable withcommercially available therapeutic recombinant Human Insulinformulations.

Thus, the process has been optimized at multiple steps, which hascumulative effects and resulted in increased yields. To name some of themajor parameters optimized in this system, the “codon-pair sequence” ofthe entire coding region, stability of mRNA, multiple copy insertions,optimized media components and growth and induction parameters.

Integration of the expression cassette into the host genome ensuresperformance and stability of the recombinant strain after repeatedsub-culturing. The possibility of multi-copy gene expression in thePichia system makes it feasible to exploit the expression, folding andsecretory capacities of the cells to the maximum. Expression of HumanInsulin as a single chain protein enables proper disulphide bridgeformation resulting in proper folding leading to a molecule that isbiologically active. Further, the process of the present disclosure inwhich in-vitro processing and use of hazardous chemicals are kept to aminimum is ideally suited for scale-up and commercial production ofrecombinant Human Insulin. The fermentation yields are significantlybetter than reported literature and unreported market figures.

In still another embodiment of the present disclosure, fermentationyields are high.

In still another embodiment of the present disclosure, use of highefficiency synthetic polymeric resins for capturing and purificationprocess resulted in enhanced recovery and purity with minimal unitoperations, as depicted in examples given below. Use of synthetic resinsenhanced the robustness of the process, stringent sanitation protocolsand ease of scale-up & overall techno-economic feasibility of theprocess.

The disclosure is further elaborated with the help of followingexamples. However, these examples should not be construed to limit thescope of disclosure.

EXAMPLES Example 1 Gene Construction and Clone Generation

26 Oligonucleotides [as given in SEQ 3] coding for the fusion protein“Mat-α-Insulin Precursor” fusion protein were designed and customsynthesized. These oligonucleotides were assembled by assembly PCR. ThePCR product is double digested with restriction enzymes BamHI and NotI(FIG. 04) and ligated into similarly processed vector pPIC9K (FIG. 03)using T4 DNA ligase.

Assembly PCR

Master stocks of oligos resuspended in water and stored in originalvials of Bioserve and kept in −20° C. Resuspension of oligos result in100 pm/μl concentration of each oligo (1 μM=1 p mole/μl). Assembly PCRrequire 0.1 μM concentration of each oligo. 10 μl of each oligo isdiluted to 200 μl (20 times) to give 5 μM solution. 1 μl of each dilutedoligo is added to PCR master mix before assembly PCR.

TABLE 1 Reaction Mix; using Phusion High Fidelity DNA Polymerase (NEB)Kit PCR MIX (50 μl) 5X rxn buffer (Hi Fidelity) 10.0 μl 10 mM dNTPs 1.0μl Oligos 26 μl Taq 0.5 μl (1 unit/μl) MilliQ 12.5 μl Total volume 50 μl

TABLE 2 PCR Program for 1st Assembly PCR PCR MIX (50 μl) Step 1 Initial98° C. 30 sec  1 cycle denaturation Step 2 Denaturation 98° C. 10 sec 30cycles Step 3 Annealing 57° C. 30 sec Step 4 Extension 72° C. 30 secRepeat 2, 3 & 4 30 times Step 5 Final extension 72° C. 7 min  1 cycleStep 6 Final Hold  4° C. αThe product of assembly PCR is used as template for 2^(nd) PCR. Productquantity is not increased in exponential way, hence it is not checked ongel electrophoresis. The product from assembly PCR is directly used astemplate for second PCR where the assembled gene is amplified by usingAOX1 primers.

TABLE 3 Reaction Mix for amplification of Clone (2^(nd) PCR) PCR MIX(NEB) (20 μl) 5X rxn buffer (Hi Fidelity) 4.0 μl 10 mM dNTP mix 0.4 μlAOX1 Primer F (100 μM) 0.2 μl AOX1 Primer R (100 μM) 0.2 μl Template(Assembly PCR mix) 1.2 μl Phusion Taq (NEB) 0.2 μl MilliQ 13.8 μl Totalvolume 20 μl

TABLE 4 PCR Program for 2^(nd) PCR PCR MIX (50 μl) Step 1 Initial 95° C.30 sec  1 cycle denaturation Step 2 Denaturation 98° C. 10 sec 30 cyclesStep 3 Annealing 57° C. 30 sec Step 4 Extension 72° C. 30 sec Repeat 2,3 & 4 30 times Step 5 Final extension 72° C 7 min  1 cycle Step 6 FinalHold  4° C. α

Resultant Sequence

Amplicon obtained from second PCR and size of the amplicon is matchingwith the size ˜500 bp (482 bp). The product of second PCR is extractedfrom agarose gel for sequencing

The ligation mix i.e. pPIC9K vector containing the ligated gene ofinterest (insulin precursor+mat-α secretion signal) is used fortransformation into chemically competent TOP 10 E. coli strain (FIG.01). CaCl₂ was used for competent cell preparation. Transformation wasdone by heat shock method. Transformation mix was plated on LB mediumcontaining Ampicillin in order to select transformed colonies. Colonieswere obtained after incubation of plates at 37° C. for 12-14 h. Glycerolstocks of transformed cells were prepared and stored at −70° C. Plasmidfrom E. coli is prepared by the protocol from Promega Kit (Wizard plusSV minipreps DNA purification system). Recombinant pPIC9K plasmid vectoris linearized with restriction enzymes SacI/BglII/salI, purified,quantified and used for transformation into Pichia pastoris.Approximately 10 μg of the linearized plasmid DNA with insert were usedfor electroporation of electrocompetent host cells. The specificationsused for electroporation are 760 Volts/5 milli seconds in 2 mm cuvette.

The transformation mixture was incubated in 1 M sorbitol for 30 min forcells to recover and further incubated in liquid regeneration media for4 hours at 30° C. with shaking. Cells were then plated on to minimalmedia lacking histidine and containing antibiotic G418. The His⁺colonies that grew on these plates were screened by PCR by using AOX1primers (FIG. 02) PCR positive cell lines are plated on fresh RD mediumplates for further screening of high copy number lines.

Clones containing multiple copies of the gene inserted into the genomewere further screened using higher concentrations of antibiotic G418.Colonies resistant to more than 4 mg G418 are considered to contain morethan twelve copies of the gene. Such colonies were selected, grown onYPD medium and maintained as glycerol stock at −70° C.

Example 2 Expression Screening

Transformed colonies growing on RD plates with 4 mg G418 were screenedfor expression by shake flask cultures according to the Invitrogen'sPichia expression protocols. More than 100 such colonies were screenedto identify few promising clones.

Each colony to be screened was grown in 5 ml YPD in a culture tube byincubating at 30° C./230 rpm/24 hrs. The seed (1 ml) is inoculated to 50ml BMG (buffered minimum glycerol medium) in 250 ml Erlenmeyer flask andincubated at 30° C./220 rpm/24 hrs. Cells were harvested by centrifugingat 2000 g/5 minutes at room temperature. Supernatant was decanted andthe cell pellet was resuspended in 25 ml BMM (buffered minimum methanolmedium) in 150 ml baffled flasks and then allowed to grow at 30° C./200rpm for 3 days. The culture was induced with methanol to a finalconcentration of 1.0% at every 24 hrs. Samples were taken at 24 hrintervals and analyzed by HPLC and SDS-PAGE.

Colonies showing good expression were made into glycerol stocks forfurther evaluation at fermentation level.

Example 3 High Cell Density Fermentation in Low Salt Minimal Media[LSMM]

Fermentation was carried in in-situ autoclavable automated vessel(BioFlo 415, NBS) of 7 litre capacity. All parameters like agitation,gas flow rates, feeding, pH adjustments, antifoam were controlled by PIDcontroller.

The fermentation medium used is a Low Salt Minimal Medium (LSMM)supplemented with trace metal salts solution (PTM4) and Biotin, asfollows:

-   -   Phosphoric acid=26.7 ml    -   CaSO₄.2H₂0=0.465 gm    -   K₂SO₄=9.1 gm    -   MgSO₄.7H₂O=7.45 gm    -   KOH=4.13 gm    -   Glycerol=50 ml    -   [All quantities per Liter of medium]

To promote rapid growth and high cell density yield in fermenter,glycerol stock is inoculated and grown in YPD medium by shake flaskculture for 18-20 hrs at 220 rpm/30° C. till OD₆₀₀ reaches 10-12. Thefirst seed is again inoculated onto YPG medium and grown at abovementioned conditions. When culture reached log phase (around 20 hrs)with OD₆₀₀ around 25-30, the cells are harvested at 1500 g/5 min andsuspended in autoclaved milliQ water. Then cells are inoculated intobasal salt medium in fermenter up to OD₆₀₀ of 5.0.

Batch phase: The fermenter medium pH adjusted to 4.75 before inoculationto avoid precipitation of medium if any. Dissolved oxygen (DO) probe isalso calibrated before inoculation. Trace metal solution of 8% added tothe vessel before and after inoculation at fixed intervals. Temperatureof the culture is maintained at 30° C. Vessel aeration was maintained as1.0 VVM pure air. Initial batch phase last for 18 hrs until OD₆₀₀reaches 120-150 with an indication of DO shoot up.

Glycerol fed batch: The glycerol fed batch started with feeding of 50%glycerol containing 12% trace metal solution on exponential feed rate toachieve high cell density before induction. Temperature and pH weremaintained at 29.5° C. and 4.80. Vessel aeration was maintained as 1.0VVM air and oxygen in a ratio of 9:1.

Methanol batch: Induction of Insulin Precursor (IP) was started byfeeding 100% methanol containing 12% PTM4 trace metal solution. Initialmethanol feed was given as spikes until culture gets adapted,subsequently switched on to exponential feed. The DO spike method wasused to determine ramp of methanol feed. Methanol feed for Mut⁺ andMut^(s) clones were based on Stratton et al., (Pichia protocols, Methodsin Molecular Biology, Vol. 103). Residual methanol in the vessel iscontinuously monitored using an in-house designed methanol probe andsensor connected to the vessel. Consumption of methanol signals increasein vessel temperature which is maintained at 28.5° C. through outmethanol fed batch. Medium pH was maintained at 4.95. Vessel aerationwas maintained as 1.5 VVM due to high density with air and oxygen in aratio begins at 85:15 and ends at 40:60 with an increment/decrement of 5at every 5 hrs. During induction phase samples were analyzed at 6-hoursinterval to check growth, induction and contamination if any. Inducedprotein secreted into broth is analyzed by HPLC using 0.1%TFA/Acetonitrile solvents in C18 column. HPLC samples at 6 hourintervals showed progressive increase in protein level (FIG. 07).Fermentation samples were also analyzed by electrophoresis (SDS-PAGE) toassess the expression of insulin precursor and its increase withinduction time (FIG. 06).

Fermentation samples during induction phase are periodically checked toknow any protease activity by azocasein assay. Fermentation conditionswere optimized for high level expression of insulin precursor which ismore than 65% of total proteins present in the final sample. Resultsshowed that the total protein present in the final sample is rangingfrom 2.3 g/L with insulin precursor being 1.5 g/L.

Example 4 Harvesting of Culture and In-Situ Capturing Insulin Precursor

When the induced culture is more than 36 hrs old, it is pumped fromfermenter into hollow fiber harvesting system with 0.2μ cartridge via aperistaltic pump. The permeate contains clarified cell free broth andretentate contains concentrated cells. The retentate with cells isrecycled back to fermenter vessel along with fresh medium to maintainnormal growth and volume (FIG. 11).

The permeate containing clarified cell free broth is passed throughcolumn packed with strong cation exchanger resin, SP sepharose(methacrylic polymer with sulphopropyl functional derivatization—GigaCapS 650, Toyopearl) at pH 3.0. for protein capturing.

The column after protein binding is washed with 2 column volumes of pH3.0 Tris buffer. The Insulin precursor selectively binds to the polymermatrix and is eluted with Tris buffer at pH 8.0. The chromatographicpurity of insulin precursor is around 75% as checked on HPLC (FIG. 08)and step yield is around 90% w/w.

Example 5 In-Situ Conversion of Insulin Precursor to Human Insulin

The PIP obtained in Example 4 is converted to Human Insulin via trypsinmediated digestion and transpeptidation followed by deblocking. Insulinprecursor eluted from ion exchange column is passed through 1 kda MWCOTFF cassette and concentrated to 100-200 mg/ml and its pH is adjusted to7.3 with 1 N HCl. The concentrated PIP is mixed withO-tert-Butyl-L-theronine tert-butyl ester acetate dissolved in 1:1 v/vdimethyl sulfoxide (DMSO):Methanol. The reaction mixture is passedthrough TPCK-treated trypsin immobilized column (25 ml XK column withcooling jacket) equilibrated with 50 mM CaCl₂ and 0.5% acetic acid. Whenreaction mixture is completely loaded into the column, the column isclosed for 2-3 hrs to permit incubation of reaction contents and columntemperature is maintained at 20° C. Then the insulin precursor convertedto Insulin butyl ester is eluted and checked by HPLC (FIG. 09).

After the completion of reaction in-situ, the elute from TPCK trypsincolumn is mixed with deblocking buffer i.e. 0.1% tryptophan intrifluoroacetic acid (TFA), incubated for 20 min at room temperature andpassed into into hydrophobic interaction chromatography column (PPG 650M Toyopearl) which is equilibrated with 100 mM Acetic acid having 0.8 MAmmonium sulphate for binding. The column is then washed with 100 mMAcetic acid with 0.4 M Ammonium sulphate, for 2 column volumes. Finallythe bound Insulin was eluted with 100 mM acetic acid and lyophilized.

Step yield=75%Chromatographic purity=85%

Example 6 Final Polishing of Human Insulin

Human Insulin obtained in example 5 is further purified from smallmolecular weight impurities and salts by size exclusion chromatographycolumn packed with sepahdex G25 matrix and checked by HPLC andlyophilized to powder form.

Step yield=85%Chromatographic purity=98.5%

The purified Human Insulin meets the quality norms as per monograph ofrecombinant Human Insulin under British Pharmacopoeia 2007, by HPLCanalysis (FIG. 10) SDS PAGE (15%) (FIG. 13) and western blot (FIG. 14)of purified human insulin and comparison with commercial formulation isprovided and.

1) A polynucleotide sequence as set forth in SEQ ID NO:
 2. 2) Thesequence as claimed in claim 1, wherein the polynucleotide encodes afusion polypeptide comprising recombinant Human Insulin Precursor andsignal peptide. 3) A polypeptide sequence as set forth in SEQ ID NO: 1.4) The sequence as claimed in claim 3, wherein the polypeptide is afusion polypeptide comprising recombinant Human Insulin Precursor andsignal peptide. 5) The sequence as claimed in claim 3, wherein thepolypeptide sequence corresponds to polynucleotide sequence set forth inSEQ ID NO: 2, wherein the polynucleotide is subjected topost-transcriptional modification and codon optimization to obtaincorresponding polypeptide of SEQ ID NO:
 1. 6) A method for obtainingrecombinant insulin precursor molecule having polypeptide sequence asset forth in SEQ ID NO: 1, said method comprising steps of: a)synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 bycombining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR,and inserting the synthesized sequence in a vector, b) transforming ahost cell with said vector followed by antibiotic screening hostselection, and c) fermenting the selected transformed host cell andin-situ capturing of the insulin precursor molecule to obtain saidprecursor having polypeptide sequence as set forth in SEQ ID NO:
 1. 7)The method as claimed in claim 6, wherein the polypeptide is a fusionpolypeptide comprising recombinant Human Insulin Precursor and signalpeptide. 8) The method as claimed in claim 6, wherein the synthesizedpolynucleotide and the vector are subjected to restriction enzymedigestion for insertion of the polynucleotide into the expression vectorand wherein restriction enzymes are selected from a group comprisingBamHI, NotI, SacI, BgIII and SacII or any combination thereof. 9) Themethod as claimed in claim 6, wherein the vector is selected from agroup comprising pPIC9K and pPICZα, preferably pPIC9K and wherein thehost is selected from a group comprising Pichia pastoris, Pichiamethanolica, Pichia guilliermondii and Pichia caribbica, preferablyPichia pastoris. 10) The method as claimed in claim 6, wherein thein-situ capturing of the precursor molecule is carried out by hollowfibre harvesting system and ion-exchange chromatographic column toobtain said precursor. 11) A method of downstream processing for in-situcapturing of protein precursor molecule during fermentation process,said method comprising steps of: a) simultaneous pumping of fermentationproduct obtained during fermentation into a hollow fibre harvestingsystem to obtain permeate and retentate, b) recycling of the retentateinto the fermentor, and c) subjecting the permeate through ion-exchangechromatographic column followed by washing with TRIS elution buffer toobtain said protein precursor molecule. 12) The method as claimed inclaim 11, wherein the permeate comprise of clarified cell free broth andthe retentate comprise of concentrated cells. 13) The method as claimedin claim 11, wherein the retentate is recycled back to fermentor vesselalong with fresh medium in the fermentor and the permeate is passedthrough the ion-exchange chromatographic column for capturing theprotein precursor. 14) The method as claimed in claim 11, wherein theprotein precursor selectively binds to polymer matrix of theion-exchange chromatographic column and is eluted with the elutionbuffer. 15) A method of downstream processing for in-situ conversion ofprotein precursor molecule into functional protein molecule, said methodcomprising step of: a) concentrating the precursor molecule through TFFCassette and mixing the concentrate with organic solution to obtainretentate reaction mixture, b) subjecting the reaction mixture toincubation through TPCK trypsin immobilized column to obtain proteinester, and c) subjecting the ester to deblocking buffer followed byhydrophobic interaction chromatographic column to obtain said functionalprotein molecule. 16) The method as claimed in claim 15, wherein theprecursor molecule is concentrated to a range of about 100 mg/ml toabout 200 mg/ml. 17) The method as claimed in claim 15, wherein theorganic solution comprise of O-tert-Butyl-L-theronine tert-butyl esteracetate dissolved in 1:1 v/v dimethyl sulfoxide (DMSO):Methanol and thedeblocking buffer comprise a combination of tryptophan andtrifluoroacetic acid. 18) The method as claimed in claim 15, wherein theTPCK column is equilibrated with a combination of CaCl₂ and Acetic acidand the hydrophobic interaction chromatographic column is equilibratedwith a combination of Acetic acid and Ammonium sulphate. 19) The methodas claimed in claim 15, wherein time for the incubation ranges fromabout 1.5 hrs to about 3.5 hrs and temperature for the incubation rangesfrom about 15° C. to about 25° C. 20) A method for obtaining recombinantinsulin molecule from a precursor molecule having polypeptide sequenceas set forth in SEQ ID NO: 1, said method comprising steps of: a)synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 bycombining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR, b)inserting the synthesized sequence in a vector and transforming a hostcell with said vector followed by antibiotic screening host selection,c) fermenting the selected transformed host cell followed by downstreamprocessing for in-situ capturing of the insulin precursor molecule, andd) in-situ conversion of insulin precursor molecule having polypeptidesequence as set forth in SEQ ID NO: 1 into said recombinant insulinmolecule. 21) The method as claimed in claim 20, wherein the polypeptideis a fusion polypeptide comprising recombinant Human Insulin Precursorand signal peptide. 22) The method as claimed in claim 20, wherein thesynthesized polynucleotide and the vector are subjected to restrictionenzyme digestion for insertion of the polynucleotide into the expressionvector and wherein the restriction enzymes are selected from a groupcomprising BamHI, NotI, SacI, BgIII and SacII or any combinationthereof. 23) The method as claimed in claim 20, wherein the vector isselected from a group comprising pPIC9K and pPICZα, preferably pPIC9Kand wherein the host is selected from a group comprising Pichiapastoris, Pichia methanolica, Pichia guilliermondii and Pichiacaribbica, preferably Pichia pastoris. 24) The method as claimed inclaim 20, wherein the in-situ capturing of the precursor molecule iscarried out by hollow fibre harvesting system and ion-exchangechromatographic column to obtain said precursor. 25) The method asclaimed in claim 20, wherein the in-situ conversion of the precursormolecule is carried out by subjecting the precursor molecule to TFFCassette and TPCK trypsin immobilized column to obtain protein ester.26) The method as claimed in claim 25, wherein the protein ester issubjected to deblocking buffer followed by hydrophobic interactionchromatographic column to obtain said recombinant insulin molecule. 27)A recombinant vector comprising polynucleotide sequence set forth in SEQID NO:
 2. 28) The vector as claimed in claim 27, wherein the vector isselected from a group comprising pPIC9K and pPICZα, preferably pPIC9K.29) A recombinant host cell, transformed by introduction of a vectorcomprising polynucleotide sequence set forth in SEQ ID NO:
 2. 30) Thehost cell as claimed in claim 29, wherein the host is selected from agroup comprising Pichia pastoris, Pichia methanolica, Pichiaguilliermondii and Pichia caribbica, preferably Pichia pastoris andwherein the vector is selected from a group comprising pPIC9K andpPICZα, preferably pPIC9K.